The role that RNA molecules might be playing in modulating eukaryotic gene expression has moved to center stage. It has recently been discovered that two small regulatory RNAs, lin-4 RNA and let-7 RNA, which control the timing of cell divisions during C. elegans development, are members of a large class of 21- to 24-nt noncoding RNAs, called microRNAs (miRNAs). Thus far, dozens of miRNA genes have been reported in C. elegans, Drosophila, and humans. This proposal focuses on the genomics and functional genomics of this newly identified class of genes, with the broad, long-term objective of understanding the roles of RNA in regulating gene expression. The specific aims are: 1) to use molecular methods to identify additional miRNA genes, 2) to develop and apply bioinformatic tools for identifying miRNA genes, and 3) to examine the consequence of disrupting miRNA genes or functions.Experiments of Aims #1 and #2 will identify and examine the expression of hundreds of miRNA genes, including most of the miRNA genes in nematodes and fish. They will also detect mouse and human candidate genes. Thus, they will greatly expand the annotation of miRNA genes in the databases-a resource for all biologists, and of particular value for those concerned with eukaryotic gene regulation, development, and disease. Experiments of Aim #1 will also search for miRNAs in plants and fungi, in an effort to expand the known phylogenetic distribution of these noncoding RNAs. Experiments of Aim #3 will attempt to knockout 100 miRNA genes in C. elegans. They will also explore methods for inhibiting miRNA function in zebrafish. The phenotypes of miRNA disruptions will provide key insights as to which regulatory networks include miRNAs and what fraction of miRNAs have discernable, non-redundant roles in development, differentiation and other processes. Because so little is known about this newly identified class of genes, speculation is rife on the processes in which they might function in humans. Identifying the miRNA genes and surveying their functions in model organisms will provide important insights and reagents for understanding the roles of miRNAs in humans and learning how their dysfunction might contribute to disease.